Figure 4. Viability and selectivity of the engineered CreAAF interface.

(a) Cre-Lox recombination assay. 13 bp repeats are indicated in cyan (220 bp) and magenta (34 bp), while the 8 bp spacers are indicated in yellow. Recombination swaps the 13 bp repeats. ³²P-5′-labelled 220 bp Lox-containing restriction fragments (*) were reacted with Cre proteins and 34 bp synthetic Lox (2:1 Cre monomers:Lox site) yielding 141 bp and 113 bp products and HJ intermediates. DNA components were separated by electrophoresis through SDS-PAGE gels, then visualized and quantified by phosphorimaging.

(b) CreAAF is competent for LoxP x LoxP recombination. The concentration-dependences of active complex assembly as indicated by substrate turnover levels were compared for CreWT and CreAAF reactions. The concentrations of 2:1 Cre:LoxP complexes are indicated above each lane. The relevant complexes are diagrammed on the left of the corresponding phosphorimages. CTD and CTH interface surfaces are indicated as described in Figure 3b.

(c) Quantification of LoxP x LoxP titration reactions. Averaged, normalized measurements from 2–4 independent titration experiments and their standard deviations are shown by data points and error bars. The isotherms were generated from averaged fit Hill binding parameters given in Table 1³¹. CreAAF reactions achieved comparable maximal levels to CreWT, but required two-fold higher complex concentrations to achieve 50% maximum turnover.

(d) CreAAF discriminates against wildtype CTH-CTD interfaces. In an “interference” assay, CreAAF and CreALSHG were recruited to adjacent positions on chimeric LoxPM7 sites (bottom left) and their ability to form active complexes was assessed, compared to a heterotetramer containing only wildtype interfaces (CreWT+CreALSHG/LoxPM7, upper left). Reactions were performed as described in (a) and (b).

(e) Quantification of the interference assay. Titration data from (d) were treated as in (c). Data points and error bars depict the normalized averages and their standard deviations for 3 or 4 experiments. The isotherms are calculated from the averaged Hill parameters given in Table 1. CreAAF is less efficient at forming heterodimers with CreALSHG on chimeric LoxPM7 sites compared to CreWT, as evidenced by an increased S_0.5 value and reduced substrate turnover level compared to CreWT+CreALSHG reactions.